Human kinase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application claims the benefit of U.S. ProvisionalApplication No. 60/282,031, which was filed on Apr. 6, 2001, and isherein incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of a novel human polynucleotide encoding a protein thatshares sequence similarity with animal kinases. The inventionencompasses the described polynucleotide, host cell expression systems,the encoded protein, fusion proteins, polypeptides and peptides,antibodies to the encoded protein and peptides, and geneticallyengineered animals that either lack or overexpress the disclosedpolynucleotide, antagonists and agonists of the protein, and othercompounds that modulate the expression or activity of the proteinencoded by the disclosed polynucleotide, which can be used fordiagnosis, drug screening, clinical trial monitoring, the treatment ofdiseases and disorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given their physiological importance,kinases have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of a nucleotide that encodes a novel human protein, andthe corresponding amino acid sequence of this protein. The novel humanprotein (NHP), described for the first time herein, shares structuralsimilarity with animal kinases, including, but not limited to,serine/threonine kinases, calcium/calmodulin dependent kinases, andmyosin light chain kinases. As such, the novel polynucleotide encodes anew kinase protein having homologues and orthologs across a range ofphyla and species.

The novel human polynucleotide described herein (SEQ ID NO:1) encodes anopen reading frame (ORF) encoding a protein of 385 amino acids in length(SEQ ID NO:2).

The invention also encompasses agonists and antagonists of the describedNHP, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with the native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHP (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHP (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof the described NHP. When the unique NHP sequence described in SEQ IDNO:1 is “knocked-out” it provides a method of identifying phenotypicexpression of the particular gene, as well as a method of assigningfunction to previously unknown genes. In addition, animals in which theunique NHP sequence described in SEQ ID NO:1 is “knocked-out” provides aunique source in which to elicit antibodies to homologous andorthologous proteins, which would have been previously viewed by theimmune system as “self” and therefore would have failed to elicitsignificant antibody responses. To these ends, gene trapped knockout EScells have been generated in murine homologs of the described NHP.

Additionally, the unique NHP sequence described in SEQ ID NO:1 is usefulfor the identification of protein coding sequences, and mapping a uniquegene to a particular chromosome. This sequence identifies biologicallyverified exon splice junctions, as opposed to splice junctions that mayhave been bioinformatically predicted from genomic sequence alone. Thesequence of the present invention is also useful as an additional DNAmarker for restriction fragment length polymorphism (RFLP) analysis, andin forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists of, NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHP and/or NHP products, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of a novel human ORF thatencodes the described novel human kinase protein.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHP described for the first time herein, is a novel protein that isexpressed in, inter alia, human cell lines, and human fetal brain, lymphnode, trachea, kidney, testis, fetal kidney, fetal lung, and 6-week oldembryo cells. The described sequences were compiled from human genomicsequence and cDNAs made from human testis, kidney, lymph node, trachea,and fetal brain mRNAs (Edge Biosystems, Gaithersburg, Md., Clontech,Palo Alto, Calif.).

The present invention encompasses the nucleotide presented in theSequence Listing, host cells expressing such a nucleotide, theexpression product of such a nucleotide, and: (a) nucleotides thatencode mammalian homologs of the described polynucleotide, including thespecifically described NHP, and the NHP products; (b) nucleotides thatencode one or more portions of the NHP that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences, including, but not limited to, the novel regions of anyactive domain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHP in which all ora part of at least one domain is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences, including, but notlimited to, soluble proteins and peptides in which all or a portion ofthe signal sequence is deleted; (d) nucleotides that encode chimericfusion proteins containing all or a portion of the coding region of theNHP, or one of its domains (e.g., a receptor/ligand binding domain,accessory protein/self-association domain, etc.) fused to anotherpeptide or polypeptide; or (e) therapeutic or diagnostic derivatives ofthe described polynucleotide, such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing.

As discussed above, the present invention includes the human DNAsequence presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species, and mutant NHPS, whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, or directedevolution as described in, for example, U.S. Pat. Nos. 5,837,458 or5,723,323, both of which are herein incorporated by reference). Theinvention also includes degenerate nucleic acid variants of thedisclosed NHP polynucleotide sequence.

Additionally contemplated are polynucleotides encoding a NHP ORF, or itsfunctional equivalent, encoded by polynucleotide sequences that areabout 99, 95, 90, or about 85 percent similar to corresponding regionsof SEQ ID NO:1 (as measured by BLAST sequence comparison analysis using,for example, the GCG sequence analysis package, as described herein,using default parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP-encoding polynucleotide. Such hybridization conditions canbe highly stringent or less highly stringent, as described herein. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80 bases long, or about 34 to about 45 baseslong, or any variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a microarray or high-throughput “chip” format).Additionally, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used to represent all or a portion of thedescribed NHP sequence. An oligonucleotide or polynucleotide sequencefirst disclosed in at least a portion of the sequence of SEQ ID NO:1 canbe used as a hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).Of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in thesequence of SEQ ID NO:1, or an amino acid sequence encoded thereby.Methods for attaching biopolymers to, or synthesizing biopolymers on,solid support matrices, and conducting binding studies thereon, aredisclosed in, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752,5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and4,689,405, the disclosures of which are herein incorporated by referencein their entirety.

Addressable arrays comprising sequences first disclosed in SEQ ID NO:1can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. Preferably the probes consist of 60nucleotides, and more preferably 25 nucleotides, from the sequence firstdisclosed in SEQ ID NO:1.

For example, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described NHP sequence. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, such polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within the nucleotide sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions, andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1 and/or 2 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1 and/or 2can also be used in the identification, selection, and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets, andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

As an example of utility, the sequences first disclosed in SEQ ID NOS:1and/or 2 can be utilized in microarrays, or other assay formats, toscreen collections of genetic or other biologic material from patientswho have a particular medical condition. These investigations can alsobe carried out using the sequences first disclosed in SEQ ID NOS:1and/or 2 in silico, and by comparing previously collected geneticdatabases and the disclosed sequences using computer software known tothose in the art.

Thus the sequences first disclosed in SEQ ID NOS:1 and/or 2 can be usedto identify mutations associated with a particular disease, and also indiagnostic or prognostic assays.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence, inconjunction with the presence of one or more specific oligonucleotidesequence(s) first disclosed in SEQ ID NO:1. Alternatively, a restrictionmap specifying the relative positions of restriction endonucleasedigestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHP antisense molecules, useful, for example, in NHP generegulation and/or as antisense primers in amplification reactions of NHPnucleic acid sequences. With respect to NHP gene regulation, suchtechniques can be used to regulate biological functions. Further, suchsequences can be used as part of ribozyme and/or triple helix sequencesthat are also useful for NHP gene regulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupincluding, but not limited to, a phosphorothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

Low stringency conditions are well-known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (and periodic updates thereof), and Ausubel et al.,1989, supra.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, genomic DNA and/or cDNA obtained byreverse transcription of mRNA prepared from, for example, human ornon-human cell lines or tissue, such as kidney or testis, known toexpress, or suspected of expressing, an allele of a NHP gene.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known to express, orsuspected of expressing, a NHP gene). A reverse transcription (RT)reaction can be performed on the RNA using an oligonucleotide primerspecific for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” using a standard terminal transferase reaction, the hybrid maybe digested with Rnase H, and second strand synthesis may then be primedwith a complementary primer. Thus, cDNA sequences upstream of theamplified fragment can be isolated. For a review of cloning strategiesthat can be used, see, e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown to express, or suspected of expressing, a NHP, in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well-known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPgene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of carrying, or known to carry, a mutantNHP allele (e.g., a person manifesting a NHP-associated phenotype suchas, for example, immune disorders, obesity, high blood pressure, etc.),or a cDNA library can be constructed using RNA from a tissue known toexpress, or suspected of expressing, a mutant NHP allele. A normal NHPgene, or any suitable fragment thereof, can then be labeled and used asa probe to identify the corresponding mutant NHP allele in suchlibraries. Clones containing mutant NHP sequences can then be purifiedand subjected to sequence analysis according to methods well-known tothose skilled in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.).

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies tothe NHP are likely to cross-react with a corresponding mutant NHPexpression product. Library clones detected via their reaction with suchlabeled antibodies can be purified and subjected to sequence analysisaccording to methods well-known in the art.

An additional application of the described novel human polynucleotidesequence is its use in the molecular mutagenesis/evolution of proteinsthat are at least partially encoded by the described novel sequenceusing, for example, polynucleotide shuffling or related methodologies.Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458,which are herein incorporated by reference in their entirety.

The invention also encompasses: (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators, and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

Where, as in the present instance, some of the described NHP peptides orpolypeptides are thought to be cytoplasmic, expression systems can beengineered that produce soluble derivatives of the NHP (corresponding toNHP extracellular and/or intracellular domains, or truncatedpolypeptides lacking one or more hydrophobic domains) and/or NHP fusionprotein products (especially NHP-Ig fusion proteins, i.e., fusions of aNHP domain to an IgFc), NHP antibodies, and anti-idiotypic antibodies(including Fab fragments) that can be used in therapeutic applications.Preferably, the above expression systems are engineered to allow thedesired peptide or polypeptide to be recovered from the culture media.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of theNHP, as well as compounds or nucleotide constructs that inhibitexpression of a NHP sequence (transcription factor inhibitors, antisenseand ribozyme molecules, or open reading frame sequence or regulatorysequence replacement constructs), or promote the expression of a NHP(e.g., expression constructs in which a NHP coding sequence isoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

The NHP or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs or inappropriately expressed NHPs for the diagnosis ofdisease. The NHP protein or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of theNHP in the body. The use of engineered host cells and/or animals canoffer an advantage in that such systems allow not only for theidentification of compounds that bind to an endogenous receptor/ligandof the NHP, but can also identify compounds that trigger NHP-mediatedactivities or pathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives, such as NHP peptides/domains corresponding to theNHP, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of the NHP, or a domain of the NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway), can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of a soluble NHP, a NHP-IgFc fusion protein, or ananti-idiotypic antibody (or its Fab) that mimics the NHP, could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of the NHP, a NHP peptide, or a NHPfusion protein to the body. Nucleotide constructs encoding functionalNHPs, mutant NHPs, as well as antisense and ribozyme molecules can alsobe used in “gene therapy” approaches for the modulation of NHPexpression. Thus, the invention also encompasses pharmaceuticalformulations and methods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The nhp sequences

The cDNA sequence (SEQ ID NO:1) and the corresponding deduced amino acidsequence (SEQ ID NO:2) of the described NHP are presented in theSequence Listing. The NHP nucleotide sequence was obtained from cDNAsobtained using probes and/or primers generated from human genomicsequence.

A number of polymorphisms that may occur in the described NHP wereidentified, including: a G/T polymorphism at the nucleotide positionrepresented by position 79 of SEQ ID NO:1, which can result in an ala orthr at the region corresponding to amino acid (aa) position 27 of SEQ IDNO:2; an A/G polymorphism at nucleotide position 647 of SEQ ID NO:1,which can result in an asp or gly at aa position 216 of SEQ ID NO:2; aG/A polymorphism at nucleotide position 660 of SEQ ID NO:1, both ofwhich result in a lys at aa position 220 of SEQ ID NO:2; and a C/Tpolymorphism at nucleotide position 873 of SEQ ID NO:1, both of whichresult in a his at aa position 291 of SEQ ID NO:2. The present inventioncontemplates sequences incorporating any or all of the abovepolymorphisms, as well as all permutations and combinations thereof.

Expression analysis has provided evidence that the described NHP isexpressed in a variety of tissues, and that the NHP shares significantsimilarity with a variety of protein kinases. Given the physiologicalimportance of protein kinases, they have been subject to intensescrutiny, as exemplified and discussed in U.S. Pat. Nos. 5,756,289 and5,817,479, herein incorporated by reference in their entirety, whichadditionally-describe a variety of uses and applications for thedescribed NHP.

The described NHP can be used to map the coding region of thecorresponding human genomic locus (chromosome mapping, see GENBANKaccession no. AC023194).

An additional application of the described novel human polynucleotidesequence is its use in the molecular mutagenesis/evolution of proteinsthat are at least partially encoded by the described novel sequenceusing, for example, polynucleotide shuffling or related methodologies.Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458,which are herein incorporated by reference in their entirety.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees, may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to, pronuclear microinjection(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus-mediatedgene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem cells(Thompson et al., 1989, Cell 56:313-321); electroporation of embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated genetransfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review ofsuch techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry a NHPtransgene in all their cells, as well as animals that carry a transgenein some, but not all their cells, i.e., mosaic animals or somatic celltransgenic animals. A transgene may be integrated as a single transgene,or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. Atransgene may also be selectively introduced into and activated in aparticular cell-type by following, for example, the teaching of Lasko etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell-type of interest, and will be apparent to thoseof skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particularcell-type, thus inactivating the endogenous NHP gene in only thatcell-type, by following, for example, the teaching of Gu et al., 1994,Science 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

Once transgenic animals have been generated, the expression of therecombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

The present invention also provides for “knock-in” animals. Knock-inanimals are those in which a polynucleotide sequence (i.e., a gene or acDNA) that the animal does not naturally have in its genome is insertedin such a way that it is expressed. Examples include, but are notlimited to, a human gene or cDNA used to replace its murine ortholog inthe mouse, a murine cDNA used to replace the murine gene in the mouse,and a human gene or cDNA or murine cDNA that is tagged with a reporterconstruct used to replace the murine ortholog or gene in the mouse. Suchreplacements can occur at the locus of the murine ortholog or gene, orat another specific site. Such knock-in animals are useful for the invivo study, testing and validation of, intra alia, human drug targets,as well as for compounds that are directed at the same, and therapeuticproteins.

5.2 NHP and NHP polypeptides

The NHP, NHP products, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHP, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto the NHP, and as reagents in assays for screening for compounds thatcan be used as pharmaceutical reagents useful in the therapeutictreatment of mental, biological, or medical disorders and diseases.Given the similarity information and expression data, the described NHPcan be targeted (by drugs, oligonucleotides, antibodies, etc.) in orderto treat disease, or to therapeutically augment the efficacy oftherapeutic agents.

The Sequence Listing discloses the amino acid sequence encoded by thedescribed NHP-encoding polynucleotide. The NHP has an initiatormethionine in a DNA sequence context consistent with eucaryotictranslation initiation site, and a signal-like sequence, indicating thatthe NHP can be secreted or membrane-associated.

The NHP amino acid sequence of the invention includes the amino acidsequence presented in the Sequence Listing, as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described herein are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequence presentedin the Sequence Listing, when taken together with the genetic code (see,for example, Table 4-1 at page 109 of “Molecular Cell Biology”, 1986, J.Darnell et al., eds., Scientific American Books, New York, N.Y., hereinincorporated by reference), are generically representative of all thevarious permutations and combinations of nucleic acid sequences that canencode such an amino acid sequence.

The invention also encompasses proteins that are functionally equivalentto the NHP products encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, the abilityto effect an identical or complementary downstream pathway, or a changein cellular metabolism (e.g., proteolytic activity, ion flux,phosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described herein, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where the NHP peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHP peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andstandard biochemical methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of a NHP, but to assess biological activity, e.g., incertain drug screening assays.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomiyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreignpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. A NHP coding sequence can be cloned individually into anon-essential region (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a NHP coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted sequence isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHP product in infected hosts (e.g.,see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHP nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHP gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHP coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, may be provided. Furthermore, the initiation codonshould be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for the desired processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theNHP sequences described herein can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the NHP product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the NHPproduct.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Anotherexemplary system allows for the ready purification of non-denaturedfusion proteins expressed in human cell lines (Janknecht et al., 1991,Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the sequenceof interest is subcloned into a vaccinia recombination plasmid such thatthe sequence's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺,nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect a NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of a NHP to an antibody moleculeor its Fab fragments could be used to target cells bearing a particularepitope. Attaching an appropriate signal sequence to a NHP would alsotransport a NHP to a desired location within the cell. Alternativelytargeting of a NHP or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes: A Practical Approach”, New, R.R.C., ed., OxfordUniversity Press, N.Y., and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures, which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of a NHP to a target site or desired organ, whereit crosses the cell membrane and/or the nucleus where the NHP can exertits functional activity. This goal may be achieved by coupling of a NHPto a cytokine or other ligand that provides targeting specificity,and/or to a protein transducing domain (see generally U.S. ProvisionalPatent Application Ser. Nos. 60/111,701 and 60/056,713, both of whichare herein incorporated by reference, for examples of such transducingsequences), to facilitate passage across cellular membranes, and canoptionally be engineered to include nuclear localization signals.

Additionally contemplated are oligopeptides that are modeled on an aminoacid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components which naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 Antibodies to NHP Products

Antibodies that specifically recognize one or more epitopes of a NHP,epitopes of conserved variants of a NHP, or peptide fragments of a NHP,are also encompassed by the invention. Such antibodies include, but arenot limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of a NHP. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes for the evaluation of the effect of test compounds onexpression and/or activity of a NHP expression product. Additionally,such antibodies can be used in conjunction with gene therapy to, forexample, evaluate normal and/or engineered NHP-expressing cells prior totheir introduction into a patient. Such antibodies may additionally beused in methods for the inhibition of abnormal NHP activity. Thus, suchantibodies may be utilized as a part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the NHP, a NHP peptide (e.g., one corresponding to afunctional domain of the NHP), truncated NHP polypeptides (a NHP inwhich one or more domains have been deleted), functional equivalents ofthe NHP, or mutated variants of the NHP. Such host animals may include,but are not limited to, pigs, rabbits, mice, goats, and rats, to namebut a few. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including, but not limited to,Freund's adjuvant (complete and incomplete), mineral salts such asaluminum hydroxide or aluminum phosphate, chitosan, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Alternatively, theimmune response could be enhanced by combination and/or coupling withmolecules such as keyhole limpet hemocyanin, tetanus toxoid, diphtheriatoxoid, ovalbumin, cholera toxin, or fragments thereof. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class, includingIgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridomasproducing the mAbs of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454), by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity, canbe used. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine mAb and a human immunoglobulinconstant region. Such technologies are described in U.S. Pat. Nos.6,114,598, 6,075,181 and 5,877,397 and their respective disclosures,which are herein incorporated by reference in their entirety. Alsoencompassed by the present invention is the use of fully humanizedmonoclonal antibodies, as described in U.S. Pat. No. 6,150,584 andrespective disclosures, which are herein incorporated by reference intheir entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: F(ab′)₂ fragments, which can be produced by pepsin digestionof an antibody molecule; and Fab fragments, which can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

Antibodies to the NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” the NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of the NHP to its cognatereceptor/ligand can be used to generate anti-idiotypes that “mimic” theNHP and, therefore, bind, activate, or neutralize the NHP, NHP receptor,or NHP ligand. Such anti-idiotypic antibodies, or Fab fragments of suchanti-idiotypes, can be used in therapeutic regimens involving aNHP-mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen the NHP, and thusnever been tolerized to the NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHPs (i.e., the NHP will be immunogenic in NHPknock-out animals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1 or the complement thereof.
 2. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:2.
 3. A substantially isolated proteinhaving the kinase activity of the protein shown in SEQ ID NO:2, which isencoded by a nucleotide sequence that hybridizes to entire SEQ ID NO:1under hybridization condition of 0.5 M NaHPO₄, 7% Sodium dodecyl sulfate(SDS), 1 mM EDTA at 65° C., and wash conditions of 0.1× SSC/0.1% SDS at68° C.
 4. A recombinant expression vector comprising the isolatednucleic acid molecule of claim
 2. 5. The recombinant expression vectorof claim 4, wherein said vector comprises SEQ ID NO:
 1. 6. A host cellcomprises the recombinant expression vector of claim 4.